PASK Antibody

What is PASK and why is it significant in molecular research?

PASK (PAS domain containing serine/threonine kinase) is a 1323-amino acid protein that belongs to the Protein kinase superfamily, specifically within the CAMK Ser/Thr protein kinase family. The cellular localization of PASK is predicted to be both cytoplasmic and nuclear, suggesting diverse functional roles within the cell . PASK's significance in molecular research stems from its dual functionality as a metabolic sensor and as a histone 3 kinase that regulates chromatin dynamics. Recent studies have demonstrated that PASK interacts with WDR5, a core component of histone methyltransferase complexes, indicating its involvement in epigenetic regulation . This interaction has been verified through reciprocal co-immunoprecipitation experiments followed by western blot analysis, confirming direct protein-protein binding. Understanding PASK's functions provides critical insights into how metabolic signals might influence gene expression through epigenetic mechanisms, making PASK antibodies essential tools for investigating these cellular processes.

What types of PASK antibodies are available for research applications?

PASK antibodies are available in several formats designed for different experimental applications. Commercially available antibodies include both polyclonal and monoclonal varieties, with the former providing broader epitope recognition and the latter offering greater specificity for particular protein domains . Current options include:

• Polyclonal antibodies that target specific amino acid regions of the PASK protein, including N-terminal regions (AA 1-100), middle sections (AA 425-475, AA 482-602), and C-terminal domains (AA 1194-1323) .

• Monoclonal antibodies with specific clone designations (e.g., 6D10, 6B7) that target defined epitopes, providing consistent lot-to-lot performance for long-term studies .

• Antibodies with different host origins, primarily rabbit and mouse, allowing flexibility in co-staining experiments and secondary antibody selection .

• Conjugated antibodies with reporter molecules such as HRP, FITC, or biotin for direct detection without secondary antibodies in certain applications .

The selection of an appropriate antibody should be guided by the specific research question, the experimental technique being employed, and the species of the sample being analyzed.

How should researchers validate PASK antibodies for their experimental systems?

Antibody validation is critical for ensuring reliable and reproducible results in PASK research. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines or tissues with known PASK expression patterns. HEK293T cells expressing human PASK-V5 and their corresponding vector controls can serve as excellent positive and negative controls, respectively . Additionally, PASK knockout or knockdown models provide stringent negative controls for antibody specificity testing.

• Cross-reactivity assessment: If working with multiple species, verify the antibody's reactivity across species boundaries. Some commercially available antibodies are reactive with human, mouse, and rat PASK, making them versatile for comparative studies .

• Application-specific validation: Confirm the antibody works in your specific application. While an antibody may be validated for Western blotting, its performance in immunofluorescence or immunohistochemistry may differ substantially. Test the antibody in all planned applications before proceeding with full experiments .

• Epitope mapping: Consider the epitope recognized by the antibody. Antibodies targeting different regions of PASK (N-terminal PAS domain versus C-terminal kinase domain) may yield different results depending on protein folding, post-translational modifications, or protein-protein interactions that could mask epitopes in particular cellular contexts .

• Orthogonal method verification: Confirm key findings using alternative methods or multiple antibodies targeting different PASK epitopes to strengthen confidence in the results.

What are the optimal conditions for using PASK antibodies in Western blotting?

Optimizing Western blot protocols for PASK detection requires attention to several critical parameters:

• Sample preparation: When preparing samples for PASK detection, include protease inhibitors to prevent degradation. For nuclear localized PASK, optimization of nuclear extraction protocols is essential. Use RIPA buffer with phosphatase inhibitors if studying phosphorylation status of PASK or its substrates .

• Protein loading: PASK is typically expressed at moderate levels in most tissues; therefore, loading 20-50 μg of total protein per lane is recommended for endogenous detection. For overexpression systems, 10-20 μg may be sufficient .

• Gel percentage: Given PASK's large molecular weight (approximately 150 kDa), use low percentage (6-8%) SDS-PAGE gels or gradient gels (4-12%) to facilitate proper resolution of the protein.

• Transfer conditions: Implement wet transfer at 30V overnight at 4°C for efficient transfer of large proteins like PASK. Use 0.45 μm pore size PVDF membranes rather than 0.2 μm to accommodate the large protein size.

• Blocking conditions: 5% non-fat dry milk in TBST is generally effective, but for phospho-specific antibodies, 5% BSA in TBST is preferable to prevent phosphatase activity in milk.

• Antibody dilution: Primary antibody dilutions typically range from 1:500 to 1:2000 depending on the specific antibody. Secondary antibody dilutions typically range from 1:5000 to 1:10000 .

• Incubation conditions: Incubate primary antibody overnight at 4°C to maximize specific binding while minimizing background.

• Detection controls: Include molecular weight markers to confirm band size and use GAPDH or H3 antibodies as loading controls depending on the fraction being analyzed .

How should I optimize PASK antibody use in immunofluorescence experiments?

Successful immunofluorescence experiments with PASK antibodies require careful optimization:

• Fixation method: Test both paraformaldehyde (4%, 10-15 minutes) and methanol (-20°C, 10 minutes) fixation, as PASK epitope accessibility may differ between methods. Paraformaldehyde preserves cellular structure but may mask some epitopes, while methanol enhances nuclear antigen detection but can disrupt membrane structures .

• Permeabilization: For paraformaldehyde-fixed samples, permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes. The optimal concentration may vary depending on the cell type and specific PASK antibody.

• Antigen retrieval: For difficult-to-detect epitopes, particularly in tissue sections, sodium citrate buffer (pH 6.0) heat-mediated antigen retrieval may improve signal intensity.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody, with 0.1-0.3% Triton X-100 in PBS for 1-2 hours at room temperature.

• Antibody dilution: Start with a 1:100 to 1:500 dilution range for primary antibodies reactive to PASK, then optimize based on signal-to-noise ratio .

• Incubation conditions: Incubate primary antibody overnight at 4°C in a humidified chamber to reduce non-specific binding while maximizing specific signal.

• Co-localization studies: When performing co-localization studies with other proteins, carefully select compatible antibodies raised in different host species to avoid cross-reactivity of secondary antibodies.

• Controls: Include a negative control by omitting the primary antibody, and when possible, use PASK-overexpressing cells as a positive control and PASK-knockdown cells as a specificity control .

What are essential considerations for Co-Immunoprecipitation experiments with PASK antibodies?

Co-immunoprecipitation (Co-IP) is a valuable technique for studying PASK's protein interactions, particularly with chromatin-modifying complexes. Key considerations include:

• Buffer selection: For nuclear protein interactions, use high-salt IP buffers (with approximately 100 mM NaCl) containing detergent, DTT, and protease inhibitors to maintain protein structure while solubilizing complexes .

• Antibody quantity: Use 1-2 μg of antibody per 500 μg of protein lysate as a starting point. The optimal ratio may vary depending on the specific PASK antibody's affinity.

• Pre-clearing step: Pre-clear lysates with appropriate control beads (protein A/G) to reduce non-specific binding, especially when working with complex nuclear extracts.

• Incubation conditions: For detecting transient interactions, shorter incubation times (2-4 hours) at room temperature may be preferable. For stable complexes, overnight incubation at 4°C is recommended .

• Bead selection: Choose between protein A, protein G, or mixed A/G beads based on the antibody's host species and isotype. For rabbit polyclonal PASK antibodies, protein A beads often work well .

• Washing stringency: Balance between removing non-specific interactions and maintaining specific interactions. For PASK nuclear interactions, 4-5 washes with IP wash buffer containing appropriate salt concentration are typically sufficient .

• Elution methods: For subsequent functional assays, consider native elution with excess antigenic peptide rather than denaturing elution with SDS loading buffer.

• Verification approaches: Confirm interactions by reciprocal Co-IP (pull down with antibody against the interacting protein and probe for PASK) and by saving 5-10% of input for comparison in Western blot analysis .

How can researchers investigate PASK's function as a histone 3 kinase?

Investigating PASK's role as a histone 3 kinase requires specialized experimental approaches:

• In vitro kinase assays: Set up reactions containing purified PASK (either full-length or kinase domain only), recombinant histone H3, ATP, and appropriate buffer conditions (Tris-HCl pH 8.8, DTT, KCl, MgCl₂). Use 0.5-1 μg recombinant histone H3 as substrate, and detect phosphorylation using phospho-specific antibodies against specific histone residues (e.g., H3T3, H3T6, H3S10, H3T11) .

• Kinase-dead controls: Include kinase-dead PASK mutants (e.g., K1028R hPASK-V5) as negative controls to confirm that observed phosphorylation is directly attributable to PASK's kinase activity rather than contaminating kinases .

• Inhibitor studies: Incorporate PASK-specific inhibitors (e.g., Bio E-115) at various concentrations to establish dose-dependent relationships and confirm specificity. Pre-incubate the inhibitor with PASK at 37°C for 15 minutes before adding substrate .

• Cell-based validation: After identifying potential histone targets in vitro, validate these findings in cellular contexts using PASK overexpression or knockdown approaches followed by histone phosphorylation analysis. The inducible CRISPR Tet-on system for controllable PASK activation provides a powerful tool for such studies .

• Domain mapping: Use deletion mutants of PASK (e.g., constructs containing amino acids 949-1315, 405-1315, or 200-1315) to determine which domains are essential for histone kinase activity versus protein-protein interactions .

• Mass spectrometry: For unbiased identification of phosphorylation sites, perform in vitro kinase reactions followed by mass spectrometry analysis of the histone substrate to identify all modified residues.

What methods can reveal PASK's interactions with chromatin remodeling complexes?

Investigating PASK's interactions with chromatin remodeling machinery requires multiple complementary approaches:

• Direct interaction assays: Incubate purified PASK (approximately 0.3 picomoles) with equivalent amounts of recombinant components of chromatin remodeling complexes (e.g., MLL2, ASH2, WDR5, RBBP5) in appropriate buffer conditions. Capture complexes using antibodies against PASK or epitope tags (e.g., V5), and analyze by western blotting .

• Protein complex purification: Use tandem affinity purification with tagged PASK followed by mass spectrometry to identify all associated proteins in an unbiased manner.

• Proximity labeling: Employ BioID or APEX2 proximity labeling by fusing these enzymes to PASK, allowing biotinylation of proteins in close proximity to PASK in living cells, followed by streptavidin purification and mass spectrometry identification.

• Chromatin immunoprecipitation (ChIP): Perform ChIP with PASK antibodies to identify genomic regions where PASK localizes, followed by sequencing (ChIP-seq) to create genome-wide binding profiles. Correlation with histone modification patterns can reveal functional relationships.

• Sequential ChIP (Re-ChIP): To determine if PASK and specific chromatin remodeling factors co-occupy the same genomic regions, perform sequential immunoprecipitation with antibodies against PASK followed by antibodies against MLL complex components.

• Functional validation: After identifying interactions, assess their functional significance by examining how PASK knockdown or overexpression affects the activity of the interacting complexes, such as measuring H3K4 methylation levels as an indicator of MLL complex activity .

How can subcellular localization of PASK be precisely determined in different physiological conditions?

Determining PASK's subcellular localization under various physiological conditions requires:

• Subcellular fractionation: Separate nuclear, cytoplasmic, and other cellular compartments using differential centrifugation, followed by western blotting with PASK antibodies. Include compartment-specific markers such as GAPDH (cytoplasm) and histone H3 (nucleus) to verify fractionation quality .

• Live-cell imaging: Create GFP-tagged PASK constructs (pcDNA3.1 + C-eGFP) for real-time visualization of PASK localization changes in response to stimuli. Time-lapse imaging can reveal dynamic translocation events .

• Super-resolution microscopy: For detailed localization studies, use techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED), or photoactivated localization microscopy (PALM) with appropriately validated PASK antibodies.

• Quantitative image analysis: Develop algorithms to quantify nuclear/cytoplasmic ratios of PASK immunofluorescence across multiple cells and conditions, ensuring statistical robustness.

• Stimulus-dependent localization: Examine PASK localization under various metabolic conditions (glucose deprivation, amino acid starvation, etc.) to connect its sensor function with its chromatin-associated roles.

• Co-localization analysis: Perform dual immunofluorescence with PASK antibodies and markers for specific nuclear subcompartments (nucleoli, speckles, PML bodies) to precisely map PASK's subnuclear distribution.

• Domain requirements: Use PASK deletion constructs to identify which protein domains drive localization to specific compartments under different conditions .

What approaches should be used for quantifying PASK expression or activity data?

Accurate quantification of PASK expression or activity requires rigorous analytical approaches:

• Western blot quantification: When quantifying PASK protein levels, normalize band intensity to appropriate loading controls—GAPDH for whole cell or cytoplasmic fractions and histone H3 for nuclear fractions . Use linear range detection methods and avoid saturated signals that can mask real differences.

• Immunofluorescence quantification: For cellular immunofluorescence, measure mean fluorescence intensity within defined cellular compartments across multiple cells (n>30 per condition) using consistent exposure settings. Report both mean values and measures of dispersion (standard deviation or standard error).

• Activity normalization: For kinase activity assays, normalize phosphorylation signal to total PASK protein input rather than simply reporting raw phosphorylation levels to account for differences in enzyme concentration.

• Statistical analysis: Apply appropriate statistical tests based on data distribution. For normally distributed data, use parametric tests (t-test for two conditions, ANOVA for multiple conditions); for non-normally distributed data, use non-parametric alternatives (Mann-Whitney U or Kruskal-Wallis).

• Biological replicates: Perform at least three independent biological replicates for any quantitative analysis, and use these independent values (rather than technical replicates) for statistical calculations.

• Dose-response relationships: When studying PASK inhibitors or activators, fit data to appropriate models (e.g., four-parameter logistic function) to determine IC50/EC50 values and Hill coefficients that characterize the response curve.

• Correlation analysis: When examining relationships between PASK levels and other variables (e.g., histone modification levels), calculate correlation coefficients and statistical significance to quantify the strength of associations.

How should researchers interpret conflicting results from different PASK antibodies?

When facing conflicting results from different PASK antibodies, consider these analytical approaches:

• Epitope mapping analysis: Compare the epitopes recognized by different antibodies. Antibodies targeting the N-terminal PAS domain (AA 1-100) versus the C-terminal kinase domain (AA 1194-1323) may yield different results if PASK undergoes conformational changes, processing, or if epitopes are masked by interacting proteins .

• Isoform specificity examination: Determine whether conflicting results could be explained by differential recognition of PASK splice variants or isoforms. Review RNA-seq or exon array data to identify which isoforms are present in your experimental system.

• Validation hierarchy: Establish a validation hierarchy based on antibody performance in controls. Give greater weight to results obtained with antibodies that show: (1) no signal in knockout/knockdown samples, (2) increased signal in overexpression systems, and (3) recognition of the expected molecular weight band .

• Orthogonal method verification: Validate key findings using antibody-independent methods such as targeted mass spectrometry or CRISPR tagging of endogenous PASK.

• Context-dependent interpretation: Consider whether discrepancies might reflect biological realities rather than technical artifacts. Different antibodies might preferentially recognize distinct PASK populations (differentially modified, complexed, or localized) in a context-dependent manner.

• Manufacturer communication: Contact antibody manufacturers with detailed documentation of discrepancies, as they may have additional validation data or insights not included in product documentation.

• Community standards: Report all antibodies used (including catalog numbers and lot numbers) when publishing, and explicitly acknowledge any discrepancies observed between different antibodies to advance community knowledge.

What quality control metrics should be implemented for PASK antibody-based experiments?

Implementing robust quality control metrics helps ensure reliability of PASK antibody-based experiments:

• Antibody validation documentation: Maintain detailed records of all validation experiments performed, including positive and negative controls, specific applications tested, and optimization parameters.

• Lot testing: Test each new antibody lot against a reference lot to ensure consistent performance, especially for long-term projects. Document any lot-to-lot variations observed.

• Signal-to-noise ratio measurement: For immunofluorescence or immunohistochemistry, calculate and report signal-to-noise ratios rather than merely showing representative images.

• Reproducibility assessment: Implement technical duplicates or triplicates within experiments and biological replicates across independent experiments. Calculate coefficients of variation to quantify reproducibility.

• Multiplexed detection: When possible, use multiplexed detection methods to simultaneously visualize PASK alongside known interacting partners or subcellular markers, providing internal consistency checks.

• Dynamic range determination: Establish the linear dynamic range for each antibody in each application by creating standard curves with varying amounts of input protein or cells with titratable PASK expression.

• Cross-laboratory validation: For critical findings, consider validation across different laboratory settings with different equipment and personnel to ensure robustness.

• Application-specific controls: For Co-IP experiments, include IgG controls matched to the host species and antibody isotype. For ChIP experiments, include input controls and IgG controls at appropriate dilutions .

What are emerging technologies for PASK research that involve antibody applications?

The field of PASK research is evolving with several promising technological advances:

• Proximity labeling proteomics: Integration of BioID or APEX2 proximity labeling with PASK antibody-based purification allows identification of transient or weak interactors that might be missed in traditional Co-IP experiments.

• Single-cell antibody-based techniques: Application of CyTOF (mass cytometry) or single-cell western blotting with PASK antibodies enables analysis of PASK expression heterogeneity within populations.

• Spatial transcriptomics integration: Combining PASK immunofluorescence with spatial transcriptomics provides insights into how PASK's chromatin regulatory functions influence local gene expression patterns.

• Conformation-specific antibodies: Development of antibodies that specifically recognize active versus inactive PASK conformations would facilitate studies of PASK activation dynamics.

• Nanobody development: Engineering of camelid-derived single-domain antibodies (nanobodies) against PASK offers advantages for live-cell imaging and potentially for targeted protein degradation approaches.

• Intrabodies: Expression of antibody fragments within cells to track or modulate PASK function in real-time without fixation or permeabilization requirements.

• Expanding orthogonal validation approaches: Combining PASK antibody-based methods with CRISPR knock-in of epitope tags or fluorescent proteins at the endogenous locus provides powerful validation strategies that the field should increasingly adopt.